The current blood supply is considerably smaller than the need therefor. Stored blood is considered unusable after about 5-6 weeks of steady deterioration in storage as determined by the inability of such cells to survive in the circulation after transfusion, which in part is caused by hemoglobin oxidation and degradation and adenosine triphosphate (ATP) depletion. Moreover, the risks involved in receiving blood from nonautologous donors remains significant. In order to address current needs, blood storage techniques must be simple, inexpensive and long-term.
Red blood cells (RBCs) survive for about 4 months under conditions of turbulent flow in the body without protein synthesis. Oxygen (O.sub.2) is essential for the conversion of hemoglobin (Hb) to met-Hb, the breakdown of which produces toxic products such as hemichrome, heroin and free Fe.sup.3+. Together with O.sub.2, these products catalyze the formation of hydroxyl radicals (OH.cndot.), and both OH.cndot. and the met-Hb breakdown products damage the red cell lipid membrane, the membrane skeleton, and the cell contents. As will be discussed hereinbelow, current approaches to red cell preservation do not address the hemoglobin breakdown damage pathway.
Refrigeration reversibly disables the enzymes essential for met-Hb reduction in vivo, increases the solubility of damaging O.sub.2 (almost by a factor of 2) in the environment of the red blood cells, and permits the level of ATP to decrease by diminishing the glycolytic rate (at 4.degree. C. the rate is about 1% of that found at 37.degree. C.). Reduction of red cell ATP concentration results in echinocyte (an unstable form of red blood cells) formation, increased rates of membrane vesiculation, loss of red cell surface area, and accelerated sequestration by splenic macrophages. Vesiculation continues throughout the cold storage period, is exacerbated by echinocyte formation, and decreases red blood cell survival by decreasing red blood cell membrane area.
The effects of elevation and preservation of ATP levels in blood storage situations has been studied. For example, in "Studies In Red Blood Cell Preservation-7. In Vivo and in Vitro Studies With A Modified Phosphate-Ammonium Additive Solution," by Greenwalt et al., Vox Sang 65, 87-94 (1993), the authors determined that the experimental additive solution (EAS-2) containing in mM: 20 NH.sub.4 Cl, 30 Na.sub.2 HPO.sub.4, 2 adenine, 110 dextrose, 55 mannitol, pH 7.15, is useful in extending the storage shelf-life of human RBCs from the current standard of 5-6 weeks to an improved standard of 8-9 weeks. Packed RBCs are suitable for transfusion following the removal of the supernatant with a single washing step. Greenwalt et al. also conclude that factors other than ATP concentration appear to play an increasingly important role in determining RBC viability after 50 days of storage. They cite the results of L. Wood and E. Beutler in "The Viability Of Human Blood Stored In Phosphate Adenine Media," Transfusion 7, 401-408 (1967), find in their own experiments that the relationship between ATP concentration and 24-hour RBC survival measurements appears to become less clear after about 8 weeks of storage. E. Beutler and C. West restate that the relationship between red cell ATP concentration and viability is a weak one after prolonged periods of storage in "Storage Of Red Cell Concentrates In CPD-A2 For 42 and 49 Days," J. Lab. Clin. Med. 102, 53-62 (1983).
In "Effects Of Oxygen On Red Cells During Liquid Storage at +4.degree. C.," by Hogman et al., Vox Sang 51, 27-34 (1986), the authors discuss that red cell content of ATP is slightly better maintained at anaerobic than at aerobic storage after 2-3 weeks. Venous blood was refrigerated and deprived of additional oxygen during storage, by placing the oxygen-permeable storage bags in a nitrogen environment and thereby gradually reducing the level of oxygen saturation. The reduction in oxygen concentration occurs slowly during storage at 4.degree. C., and is far from complete, starting at .about.60% and reaching .about.30% hemoglobin saturation at 5 weeks. No conclusion could be drawn concerning the effects of this procedure on the overall quality of stored cells. These authors did not address or significantly reduce the oxygen-dependent damage to hemoglobin and the oxygen-mediated damage caused by hemoglobin breakdown products.
Accordingly, it is an object of the present invention to provide a procedure for blood storage which addresses the problems of hemoglobin degradation, red blood cell lysis (hemolysis) and ATP depletion in a manner consistent with the practice of autologous transfusion and enhanced heterologous transfusion logistics, and which achieves significant prolongation of the time during which refrigerated storage of red blood cells is not detrimental to their subsequent use.
Another object of the present invention is to provide a procedure for prolonged blood storage while minimizing the complexity of the procedures required for preparing transfusible samples.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.